Structure and Function of the Human PON1 Polymorphism

描述（由申请人提供）：拟议研究的主要目的是了解人类PON 1基因遗传变异的生理后果，并确定导致个体间血浆PON 1水平差异较大的因素。E.大肠杆菌PON 1的表达和纯化方案为表征序列变体对PON 1催化效率的影响提供了快速的手段，同时为毒理学实验和结构/功能研究提供了充足的重组PON 1蛋白。重组人PON 1治疗急性OP暴露病例的治疗潜力的证明为拟议研究的具体目标1和2提供了动力。本申请中提出的研究有5个具体目标。Specific Aim 1的目标是改进在大肠杆菌中产生天然和变体人PON 1的表达和纯化方案。coli表达系统。具体目标2的目标是产生具有足够催化效率的PON 1变体，以防止暴露于磷酸三甲苯酯和对氧磷。具体目标3的目标是识别和验证有机磷（OP）化合物暴露的生物标志物。迫切需要暴露生物标志物，将暴露分析的时间范围延长到测量尿液或血液代谢物提供的几天窗口之外。具体目标4阐述了母体PON 1状态在保护胎儿免受妊娠期OP（二氮嗪）暴露方面的重要性。迄今为止产生的数据表明，PON 1状态是重要的，在确定敏感性二嗪磷/二唑磷和毒死蜱/毒死蜱氧磷。特异性目的5检查启动子区域表观遗传甲基化对PON 1血浆水平的影响。迄今为止，我们的研究已经表征了显著影响血浆PON 1水平的启动子多态性。对大约200种新的多态性的鉴定表明，血浆PON 1水平的变异性中只有30%似乎依赖于启动子区域的多态性。对PON 1启动子区CpG甲基化的初步研究揭示了变异性，这可能解释了另一个重要的调控来源。来自转基因PON 1小鼠谱系的数据与这种额外的调节水平一致。公共卫生相关性：该项目产生的数据有助于美国环保署谈判减少二嗪农和毒死蜱的暴露。PON 1状态分析已经确定了低PON 1状态是颈动脉疾病的危险因素，并且还确定了男性帕金森病患者和对照组之间的差异。工程重组人PON 1将提供治疗神经毒剂和杀虫剂暴露的疗法。

DESCRIPTION (provided by applicant): A major objective of the proposed research is to understand the physiological consequences of genetic variability in the human PON1 gene and to identify factors that contribute to the large differences in plasma PON1 levels among individuals. Development of E. coli PON1 expression and purification protocols provides a rapid means for characterizing the effects of sequence variants on the catalytic efficiency of PON1 and at the same time provides sufficient recombinant PON1 protein for toxicology experiments and structure/function studies. Demonstration of the therapeutic potential of recombinant human PON1 for treating cases of acute OP exposure has provided the impetus for Specific Aims 1 and 2 of the proposed research. The research proposed in this application has 5 specific aims. The goal of Specific Aim 1 is to improve the expression and purification protocols for generating native and variant human PON1s in the E. coli expression system. The goal of Specific Aim 2 is to generate PON1 variants that have sufficient catalytic efficiency to protect against exposures to tricresyl phosphate and paraoxon. The goal of Specific Aim 3 is to identify and validate biomarkers for exposure to organophosphorus (OP) compounds. There is a pressing need for biomarkers of exposure that extend the time-frame of exposure analyses beyond the few day window that measuring urinary or blood metabolites provides. Specific Aim 4 addresses the importance of maternal PON1 status in protecting the fetus from OP (diazoxon) exposure during gestation. The data generated to date indicate that PON1 status is important in determining sensitivity to diazinon/diazoxon and chlorpyrifos/chlorpyrifos oxon. Specific Aim 5 examines the influence of promoter region epigenetic methylation on PON1 plasma levels. Our research to date has characterized promoter polymorphisms that significantly influence plasma PON1 levels. Identification of approximately 200 new polymorphisms has shown that only 30% of the variability of plasma PON1 levels appears to be dependent on promoter-region polymorphisms. Preliminary examination of methylation of CpGs in the PON1 promoter region revealed variability that may explain another significant source of regulation. Data from transgenic PON1 mouse pedigrees are consistent with this additional level of regulation. PUBLIC HEALTH RELEVANCE: Data generated from this project has assisted the EPA in negotiating a reduction in diazinon and chlorpyrifos exposure. The PON1 status analyses have identified low PON1 status as a risk factor for carotid artery disease and have also identified differences between male Parkinson's patients and controls. Engineering recombinant human PON1 will provide therapeutics for treating nerve agent and insecticide exposures.